The GPCR downstream effector SNAP23 regulates MVB–PM fusion. (a) Network of proteins of interest together with direct interactors with altered phosphorylation levels upon histamine (100 µM) stimulation as identified by phosphoproteomics in HeLa and HUVEC cells. Proteins of interest are depicted with a blue rim. FC, fold change. (b) Graph showing the signal intensity values of phosphorylated peptides from proteins of interest before and after stimulation with 100 µM histamine. Data represent means ± SD of two technical replicates per condition. *, P < 0.05 (P = 0.048) using Student’s two-tailed two-sample t test. (c) Western blotting analysis on SNAP23 protein expression in six different cell lines. (d) Confocal analysis of FL (GFP-SNAP23-FL) and truncated (GFP-SNAP23-CΔ9) GFP-SNAP23 (in gray)–transfected SiHa cells labeled for CD63 (red). (e) Total projection of fusion events in CD63-pHluorin SiHa cells cotransfected with SNAP23-FL or SNAP23-CΔ9 over 3 min. Pseudocolored as in Fig. 1 j. Bars, 10 µm. (f) Quantification of fusion events in CD63-pHluorin SiHa cells cotransfected with SNAP23-FL or SNAP23-CΔ9. n ≥ 10 cells per condition. (g) Confirmation of SNAP23 knockdown (KD) at the protein level in HeLa cells. (h) Effect of SNAP23 knockdown on MVB–PM fusion in HeLa cells. n ≥ 17 cells per condition. (i) Confirmation of SNAP23 and syntaxin-4 knockdown in HeLa cells at the mRNA level. Data represent means ± SD. (j) Effect of the knockdown of SNAP23 or syntaxin-4 on the fusion activity of HeLa cells. n ≥ 11 cells per condition. ctrl, nontransfected; siCTRL, control siRNA. *, P < 0.05; **, P < 0.01 using Student’s two-tailed two-sample t test. Whiskers in the box plots (f, h, and j) represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.